Chagas"" disease is characterized by a short-term acute phase, with very few clinical symptoms, and a long-term chronic phase, usually accompanied by severe gastrointestinal and/or cardic complications which result in permanent physical disability or death.
Chagas"" disease is an endemic disease caused by the flagellate Trypanosoma cruzi. In Latin America, approximately 16 to 18 million individuals are already infected and as many as 90 million individuals are at risk of infection (W.H.O., 1991). The disease is transmitted in Nature by Triatominae vectors. As a result of effective public health measures for the control of the vector in most countries. blood transfusion is quantitatively the most important form of transmission of the disease today. In Latin America, blood samples with antibodies associated with Chagas"" disease represent 1-4% of the total blood samples in major Hemocenters. More recently, Chagas"" disease has also become a major public health concern in North America, owing to the increasing number of immigrants from Latin American countries, in the last decade. Recent studies estimate that there may be in the United States approximately 100,000 Trypanosoma cruzi-infected individuals with potential risk of transmitting Chagas"" disease by blood transfusion (Hagar and Rahimtoola, 1991).
The diagnosis of acute Chagas"" disease is not a problem because of the large number of parasites in the blood. In contrast, the chronic phase is diagnosed by serological methods because of the very small number or absence of circulating parasites. This has also restricted so far the use of polymerase chain reaction (PCR) with specific primers, as the final diagnostic test of Chagas"" disease, before a major epidemiologic survey of sera from chronic patients is carried out.
The three serological methods that are currently being used in blood banksxe2x80x94indirect hemmagglutination (IHA), indirect immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA)xe2x80x94utilize mixtures of antigens prepared from the epimastigote form of the parasite. According to the World Health Organization (WHO), at least two positive tests of the three cited above are necessary for the diagnosis of the disease. Blood samples that are positive to only one of the three tests are classified as xe2x80x9cindeterminate or inconclusivexe2x80x9d and, in consequence, discarded. The indeterminate diagnosis is associated with 20 to 90% of all blood samples that gave one or more positive tests for Chagas"" disease, depending on the methods employed and how they are applied. This high percentage of indeterminate results represents a serious problem in blood banks, both in terms of volume of discarded blood and doubtful diagnosis of Chagas"" disease. In fact, a blood sample with a false positive test is no longer used for transfusion or isolation of cells and other blood components. Such loss of donated blood also affects the production of blood derivatives such as albumin, immunoglobulins and clotting factors which are of commercial value. Conversely, a blood sample with a false negative test is a dangerous source of contamination by the parasite.
The disadvantages of the current serological methods can be summarized as follows:
1. Low sensitivity: current methods use human sera at low dilutions, with a consequent increase in the background due to the cross-reactivity with natural antibodies and low-titer antibodies resulting from nonspecific polyclonal activation. Specific recombinant or synthetic epimastigote antigens, singly or in mixtures, are not sufficiently sensitive because they react only with a limited number of specific antibodies present in the sera of chronic Chagasic patient.
2. Low specificity: serological tests using epimastigote extracts cross-react with antigens from microbial sources other than Trypanosoma cruzi, notably Leishmania and some fungal and bacterial antigens.
The invention describes the purification of the AandT and EpEx antigens, and their use in a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA), for the accurate diagnosis of Chagas"" disease. When carried out in parallel, the results of the tests taken together provide high sensitivity and high specificity not obtainable with conventional methods described in the literature and/or which are commercially available.